Human cytochrome P450 3A4 (CYP3A4) is the most abundant and clinically relevant xenobiotic-metabolizing P450 in humans and is well known for its extreme promiscuity in substrate specificity and allosteric behavior. In addition to the metabolism of drugs and endogenous compounds, CYP3A4 is implicated in oxidation and bioactivation of pesticides, insecticides, herbicides, carcinogens and environmental pollutants. CYP3A4 can also be inhibited and activated by xenobiotics, which may lead to undesired drug-drug interactions, toxicity and human death. Despite the central role of CYP3A4 in drug metabolism, its mechanism of inhibition and activation are still poorly understood. This proposal is designed to address key issues in both areas of the CYP3A4 research and centers on our advances in the structural and functional investigation of the CYP3A4- ligand binding process. The goal of Aim 1 is to optimize the CYP3A4 inhibitor pharmacophore model that was built based on our studies of analogs of ritonavir, the most potent pharmacoenhancer marketed to date. We will accurately identify the pharmacophoric determinants using novel, rationally designed compounds, which will be further optimized based on structural and functional interpretations. The structure-based CYP3A4-specific inhibitor pharmacophore could assist in identification and early elimination of potential CYP3A4 inactivators during development of drugs and other chemicals used in agriculture and public health, as well as designing more potent pharmacoenhancers. The second part of the proposal focuses on the allosteric behavior of CYP3A4, thought to arise from the multiple substrate binding. CYP3A4 can be activated by various xenobiotics and natural dietary compounds, such as plant flavonoids, but the underlying mechanism is still under debate. Our recent findings suggest that the substrate-access channel rather than the previously identified peripheral area serves as a high affinity effector binding sie. The goal of Aim 2 is to test this hypothesis by comparing the relative importance of the peripheral and intra-channel sites in the CYP3A4 activation by flavonoid-like molecules. Using an integrated structural, biochemical and biophysical approach, we will assess how disruption of these areas affects protein dynamics, substrate binding stoichiometry, kinetics and metabolism. These studies will provide novel insights into the mechanism of CYP3A4 activation, substrate cooperativity and drug-drug interactions, which is essential for prediction/prevention of chemical toxicity and understanding interindividual differences in metabolism of drugs, dietary compounds and environmental chemicals.